Background: Exposure to n-3 polyunsaturated fatty acids (PUFA) in early life is hypothesized to offer protection against atopic disease. However, there is controversy in this area, and we have previously observed that high levels of n-3 fatty acid (FA) in colostrum are associated with increased risk of allergic sensitization. Objective: The aim of the study was to assess the relationship between FA profile in breast milk and risk of childhood atopic disease. Methods: A high-risk birth cohort was recruited, and a total of 224 mothers provided a sample of colostrum (n=194) and/or 3-month expressed breast milk (n=118). FA concentrations were determined by gas chromatography. Presence of eczema, asthma and rhinitis were prospectively documented up to 7 years of age. Results: High levels of n-3 22:5 FA (docosapentaenoic acid, DPA) in colostrum were associated with increased risk of infantile atopic eczema [odds ratio (OR)=1.66 per 1 standard deviation increase, 95% confidence interval (CI)=1.11–2.48], while total n-3 concentration in breast milk was associated with increased risk of non-atopic eczema (OR=1.60, 95% CI=1.03–2.50). Higher levels of total n-6 FA in colostrum were associated with increased risk of childhood rhinitis (OR=1.59, 95% CI=1.12–2.25). There was no evidence of associations between FA profile and risk of asthma. Conclusion: In this cohort of high-risk children, a number of modest associations were observed between FA concentrations in colostrum and breast milk and allergic disease outcomes. Further research in this area with larger sample sizes is needed.